Amine oxidase activity of ß-amyloid precursor protein modulates systemic and local catecholamine levels.

The catecholamines dopamine (DA), norepinephrine (NE) and epinephrine (E) are neurotransmitters and hormones that mediate stress responses in tissues and plasma. The expression of ß-amyloid precursor protein (APP) is responsive to stress and is high in tissues rich in catecholamines. We recently reported that APP is a ferroxidase, subsuming, in neurons and other cells, the iron-export activity that ceruloplasmin mediates in glia. Here we report that, like ceruloplasmin, APP also oxidizes synthetic amines and catecholamines catalytically (K(m) NE=0.27 mM), through a site encompassing its ferroxidase motif and selectively inhibited by zinc. Accordingly, APP knockout mice have significantly higher levels of DA, NE and E in brain, plasma and select tissues. Consistent with this, these animals have increased resting heart rate and systolic blood pressure as well as suppressed prolactin and lymphocyte levels. These findings support a role for APP in extracellular catecholaminergic clearance.

Automatic twin statistics from electron backscattered diffraction data.

A new computer code has been developed to automatically extract quantitative twin statistics from electron backscatter diffraction data. The new code is an improvement upon previous codes in that it handles materials of any crystal symmetry, type I, Type II and compound twins, and general stress states. Moreover, accuracy of the results has been greatly improved. In addition, twin statistics including number, area fraction, twin thickness and twinning dependencies on orientation, grain size and neighbourhood effects can be routinely analysed. The new code has been applied to scan data from deformed magnesium, zirconium and uranium, and can potentially be used for any twinning material for which reliable electron backscatter diffraction results can be obtained.

Role of the APP non-amyloidogenic signaling pathway and targeting alpha-secretase as an alternative drug target for treatment of Alzheimer's disease.

Alzheimer's disease (AD) is the most prevalent form of dementia, and its effective disease modifying therapies are desperately needed. Promotion of non-amyloidogenic alpha-secretase cleavage of amyloid precursor protein (APP) to release soluble sAPPalpha, based on the most widely accepted "amyloid model" as a plausible mechanism for AD treatment, is the focus of this review. Modulation of alpha-secretase or "a disintegrin and metalloprotease (ADAM)"s activity via protein kinase C (PKC), calcium ion (Ca(2+)), tyrosine kinase (TK), MAP kinase (MAPK), and hormonal signaling, which regulate catabolic processing of APP, are discussed. The inhibition of amyloidogenic processing of APP by the beta- and gamma-secretase has been considered till now a promising strategy to treat AD. But beta- and gamma-secretase inhibitors, along with the available therapeutic tools for AD, have side effects. These challenges can be circumvented to certain extent; but activation of sAPPalpha release appears to be a potential alternative strategy to reduce cerebral amyloidosis. Drug screens have been performed to identify therapeutics for AD, but an effective screening strategy to isolate activators of alpha-secretase has been rarely reported. Novel reporter-based screens targeted toward APP mRNA 5' untranslated region (UTR), followed by counter-screens to detect alpha-secretase stimulators, could be important in detecting compounds to promote sAPPalpha release and reduce amyloid beta (Abeta) buildup. The primary inflammatory cytokine interleukin-1, which stimulates APP 5'UTR-directed translation of cell-associated APP, enhances processing to sAPPalpha in astrocytes and co-activates ADAM-10/ADAM-17 through MAPK signaling; thus illustrating a novel pathway that could serve as therapeutic model for AD.

Metal specificity of an iron-responsive element in Alzheimer's APP mRNA 5'untranslated region, tolerance of SH-SY5Y and H4 neural cells to desferrioxamine, clioquinol, VK-28, and a piperazine chelator.

Iron closely regulates the expression of the Alzheimer's Amyloid Precursor Protein (APP) gene at the level of message translation by a pathway similar to iron control of the translation of the ferritin L- and H mRNAs by Iron-responsive Elements in their 5' untranslated regions (5'UTRs). Using transfection based assays in SH-SY5Y neuroblastoma cells we tested the relative efficiency by which iron, copper and zinc up-regulate IRE activity in the APP 5'UTR. Desferrioxamine (high affinity Fe3+ chelator), (ii) clioquinol (low affinity Fe/Cu/Zn chelator), (iii) piperazine-1 (oral Fe chelator), (iv) VK-28 (oral Fe chelator), were tested for their relative modulation of APP 5' UTR directed translation of a luciferase reporter gene. Iron chelation based therapeutic strategies for slowing the progression of Alzheimer's disease (and other neurological disorders that manifest iron imbalance) are discussed with regard to the relative neural toxic action of each chelator in SH-SY5Y cells and in H4 glioblastoma cells.

Pleural fluid characteristics of patients with symptomatic pleural effusion after coronary artery bypass graft surgery.

BACKGROUND: This study describes the pleural fluid characteristics of patients who develop symptomatic pleural effusions after coronary artery bypass graft surgery (CABG). METHODS: Post-CABG patients who underwent a therapeutic thoracentesis for a symptomatic pleural effusion were included unless another explanation for the pleural effusion was present. RESULTS: During the study, 71 patients (mean age, 61 years) were identified; 49 were men and 22 were women. All patients underwent internal mammary artery grafting. Early effusions (<30 days after CABG) occurred in 45 patients (63%) and late effusions (>/=30 days after CABG) developed in 26 (37%). Early effusions were bloody (median red blood cell count, 706 x 10(12)/L [706,000 mm(3)])with a high eosinophil count (median, 0.385), whereas effusions that occurred in the late period were yellow exudates with predominant lymphocytes (median, 0.68) and monocytes (median, 0.20). The mean pleural fluid level of lactate dehydrogenase was more than 3 times the upper limit of the reference range in serum in early effusions, whereas late effusions had significantly lower lactate dehydrogenase levels. CONCLUSIONS: Characteristics of early and late effusions differ significantly, suggesting a different pathogenesis of the effusions. Patients who develop a symptomatic pleural effusion after CABG should undergo a therapeutic thoracentesis; however, further investigations are warranted only in patients who have pleural fluid characteristics different from those described.

Routine measurement of pleural fluid amylase is not indicated.

BACKGROUND: The routine measurement of pleural fluid amylase is frequently recommended, but the cost-effectiveness of this procedure is unknown. METHODS: To assess the utility of routine measurement of pleural fluid amylase in evaluating pleural effusions, we measured amylase, glucose, lactate dehydrogenase, and protein levels and blood cell counts in 379 patients undergoing thoracentesis during a 22-month period from 1997 to 1999. Of these, 199 had effusions after cardiac surgery; 61, malignant; 48, transudative; 28, parapneumonic; 2, chylous; 2, rheumatoid; 1, tuberculous; and 1, from chronic pleuritis. There were 37 exudates of unknown origin. RESULTS: Measurement of pleural fluid amylase levels did not assist in determining the origin of the effusion in any of the patients. Amylase levels greater than 100 U/L (normal serum level in our laboratory is 30-110 U/L) were found in 5 (1.3%) of 379 patients: 1 patient with congestive heart failure (amylase, 173 U/L), 2 with post-cardiac surgery effusions (144 U/L and 130 U/L), 1 with pneumonia (109 U/L), and 1 with lung cancer (105 U/L). CONCLUSIONS: The routine measurement of pleural fluid amylase levels is neither clinically indicated nor cost-effective. We suggest that pleural fluid serum amylase levels be measured only if there is a pretest suspicion of acute pancreatitis, chronic pancreatic disease, or esophageal rupture.

Iron-regulatory proteins, iron-responsive elements and ferritin mRNA translation.

Iron plays a central role in the metabolism of all cells. This is evident by its major contribution to many diverse functions, such as DNA replication, bacterial pathogenicity, photosynthesis, oxidative stress control and cell proliferation. In mammalian systems, control of intracellular iron homeostasis is largely due to posttranscriptional regulation of binding by iron-regulatory RNA-binding proteins (IRPs) to iron-responsive elements (IREs) within ferritin and transferrin receptor (TfR) mRNAs. the TfR transports iron into cells and the iron is subsequently stored within ferritin. IRP binding is under tight control so that it responds to changes in intracellular iron requirements in a coordinate manner by differentially regulating ferritin mRNA translational efficiency and TfR mRNA stability. Several different stimuli, as well as intracellular iron levels and oxidative stress, are capable of regulating these RNA-protein interactions. In this mini-review, we shall concentrate on the mechanisms underlying modulation of the interaction of IRPs and the ferritin IRE and its role in regulating ferritin gene expression.

Iron-dependent regulation of the divalent metal ion transporter.

The first step in intestinal iron absorption is mediated by the H(+)-coupled Fe(2+) transporter called divalent cation transporter 1/divalent metal ion transporter 1 (DCT1/DMT1) (also known as natural resistance-associated macrophage protein 2). DCT1/DMT1 mRNA levels in the duodenum strongly increase in response to iron depletion. To study the mechanism of iron-dependent DCT1/DMT1 mRNA regulation, we investigated the endogenous expression of DCT1/DMT1 mRNA in various cell types. We found that only the iron responsive element (IRE)-containing form, which corresponds to one of two splice forms of DCT1/DMT1, is responsive to iron treatment and this responsiveness was cell type specific. We also examined the interaction of the putative 3'-UTR IRE with iron responsive binding proteins (IRP1 and IRP2), and found that IRP1 binds to the DCT1/DMT1-IRE with higher affinity compared to IRP2. This differential binding of IRP1 and IRP2 was also reported for the IREs of transferrin receptors, erythroid 5-aminolevulinate synthase and mitochondrial aconitase. We propose that regulation of DCT1/DMT1 mRNA by iron involves post-transcriptional regulation through the binding of IRP1 to the transporter's IRE, as well as other as yet unknown factors.

Rationale for the development of cholinesterase inhibitors as anti-Alzheimer agents.

Alzheimer's disease (AD) is characterized by progressive dementia caused by the loss of the presynaptic markers of the cholinergic system in the brain areas related to memory and learning and brain deposits of amyloid beta peptide (A beta) and neurofibrillary tangles (NFT). A small fraction of early onset familial AD (FAD) is caused by mutations in genes, such as the beta-amyloid precursor protein (APP) and presenilins that increase the load of A beta in the brain. These studies together with findings that A beta is neurotoxic in vitro, provide evidence that some aggregates of this peptide are the key to the pathogenesis of AD. The yield of A beta and the processing and turnover of APP are regulated by a number of pathways including apolipoprotein E, cholesterol and cholinergic agonists. Early studies showed that muscarinic agonists increased APP processing within the A beta sequence (sAPP alpha). More recently, we have presented evidence showing that some, but not all, anticholinesterases reduce secretion of sAPP alpha as well as A beta into the media suggesting that cholinergic agonists modulate A beta levels by multiple mechanisms. Herein we review the recent advances in understanding the function of cholinesterase (ChE) in the brain and the use of ChE-inhibitors in AD. We propose and support the position that the influence of cholinergic stimulation on amyloid formation is critical in light of the early targeting of the cholinergic basal forebrain in AD and the possibility that maintenance of this cholinergic tone might slow amyloid deposition. In this context, the dual action of certain cholinesterase inhibitors on their ability to increase acetylcholine levels and decrease amyloid burden assumes significance as it may identify a single drug to both arrest the progression of the disease as well as treat its symptoms. A new generation of acetyl- and butyryl cholinesterase inhibitors is being studied and tested in human clinical trials for AD. We critically discuss recent trends in AD research, from molecular and genetic to clinical areas, as it relates to the effects of cholinergic agents and their secondary effects on A beta. Finally, we examine different neurobiological mechanisms that provide the basis of new targets for AD drug development.

Optimized RNA gel-shift and UV cross-linking assays for characterization of cytoplasmic RNA-protein interactions.

Considerable interest has recently focused on defining the mechanisms involved in the regulation of gene expression at the level of mRNA stability and translational efficiency. However, the assays used to directly investigate interactions between RNA and cytoplasmic proteins have been difficult to establish, and methods are not widely available. Here, we describe a robust method for RNA electrophoretic mobility shift and UV cross-linking assays that allows rapid detection of cytoplasmic RNA-protein interactions. For added convenience to new investigators, these assays use mini-gels with an electrophoresis time of 15-20 min, enabling a high throughput of samples. The method works successfully with many different probes and cytoplasmic extracts from a variety of cell lines. Furthermore, we provide a system to optimize characterization of the RNA-protein complex and troubleshoot most assay difficulties.

Role of cytokines in the gene expression of amyloid beta-protein precursor: identification of a 5'-UTR-binding nuclear factor and its implications in Alzheimer's disease.

One of the major neuropathological characteristics of Alzheimer's disease (AD) is the brain depositions of senile plaques that are mainly composed of toxic amyloid beta-peptide (Abeta), which is generated from a family of Abeta containing precursor proteins (AbetaPP; 695-770 amino acids). The role of cytokines and growth factors has been implicated in the pathogenesis of AD. Our goal is to determine the mode of action of cytokines on the regulation of betaPP gene expression. Here we studied the effect of different cytokines on the activity of 5'-untranslated region (5'-UTR) of betaPP mRNA in human astrocytic cells (U-373). We compared betaPP-5'-UTR activity in the presence of interleukin-1 (IL-1alpha and IL-1beta), transforming growth factor (TGF-beta1) and tumor necrosis factor TNF-alpha1. The astrocytic cells, which were treated separately with these agents, were transfected with either the vector (pSV2CAT) or pSV2UTR-CAT construct containing 90 bp of AbetaPP 5'-UTR +54 to 144 bp). This region was cloned upstream of a reporter chloramphenicol acetyl transferase gene (CAT). Our results indicate that the treatment of pSV2UTR-CAT-transfected cells with either IL-1alpha, IL-1beta, TGF-beta1 or TNF-alpha1 stimulated reporter gene activity in a factor-specific manner. This was consistent with their effects on elevating AbetaPP protein levels. Transfection of the same cells with the pSV2CAT vector lacking 5'-UTR resulted in a reduced reporter gene activity with all treatments studied. DNA-gel shift experiments indicate that the 54/144 region binds to a nuclear protein(s) in a cell type specific manner. These results suggest that 5'-UTR of the AbetaPP gene can respond to the stimulation of different cytokines, which likely regulate AbetaPP transcription and translation via regulatory elements present in the AbetaPP promoter and in 5'-UTR, respectively. The characterization of AbetaPP regulatory elements, including the 5'-UTR, will accelerate the development of novel agents against new targets for AD.

Adenosine deaminase levels in nontuberculous lymphocytic pleural effusions.

OBJECTIVES: Adenosine deaminase (ADA) can aid in the diagnosis of tuberculous pleural effusions, but false-positive findings from lymphocytic effusions have been reported. We studied the ADA levels in a variety of nontuberculous lymphocytic effusions and analyzed the relationships between ADA and conventional hematologic and biochemical parameters. METHODS: One hundred six lymphocytic pleural fluid samples (lymphocyte count > 50%) were analyzed. These included post-coronary artery bypass grafting (CABG) effusions (n = 45), malignant effusions (n = 27), miscellaneous exudative effusions (n = 10), and transudative effusions (n = 24). ADA levels were determined using the Giusti method. In 22 randomly selected cases, ADA was measured again on the same sample 6 weeks later. RESULTS: The ADA level reached the diagnostic cutoff for tuberculosis (40 U/L) in only three cases (2.8%): two lymphomas and one complicated parapneumonic effusion. There was no significant correlation between effusion ADA levels and the total leukocyte (r = 0.08), differential lymphocyte (r = 0.18) or monocyte (r = - 0.18) counts. ADA levels were significantly lower in the transudative effusions (7.2 +/- 3.5 U/L) than in post-CABG (16.6 +/- 7.2 U/L), malignant (15.3 +/- 11.2 U/L), and other exudative (15.4 +/- 13.1 U/L) effusions (p < 0.001). ADA measurements were consistent when assayed 6 weeks apart (r = 0.95; p < 0.00001; coefficient of variation, 14%). CONCLUSIONS: ADA levels in nontuberculous lymphocytic effusions seldom exceeded the diagnostic cutoff for TB. Effusion ADA levels cannot be predicted from total or differential leukocyte counts. Post-CABG pleural fluids had ADA levels similar to other nontuberculous lymphocytic effusions. ADA is stable in effusion fluids, and its measurement is reproducible.

Vascular endothelial growth factor level correlates with transforming growth factor-beta isoform levels in pleural effusions.

BACKGROUND: Recent studies have demonstrated high levels of vascular endothelial growth factor (VEGF) in exudative pleural effusions and a possible etiologic role. The factors regulating VEGF accumulation in the pleural space are unknown. Transforming growth factor (TGF)-beta is a potent stimulator of VEGF expression in vitro. We hypothesized that TGF-beta induces VEGF production in pleural tissues, and, hence, the pleural fluid VEGF levels should correlate with the levels of TGF-beta in pleural fluid of different etiologies. METHODS: Seventy pleural fluid samples were analyzed. These included 20 malignant, 13 post-coronary artery bypass grafting (CABG), 8 parapneumonic, 11 miscellaneous exudative, and 18 congestive heart failure (CHF) pleural effusions. RESULTS: Pleural fluid VEGF levels showed good correlation with those of TGF-beta(1) (r = 0.58; p < 0. 0001), TGF-beta(2) (r = 0.43; p < 0.001), and lactate dehydrogenase (r = 0.65; p < 0.001). The levels of TGF-beta(1) and TGF-beta(2) also were correlated (r = 0.60; p < 0.0001). The median levels of TGF-beta(1) (2,480 pg/mL) and TGF-beta(2) (266 pg/mL) in the CHF group were significantly lower than those in the malignant (TGF-beta(1), 4,902 pg/mL; TGF-beta(2), 428 pg/mL), post-CABG (TGF-beta(1), 5,456 pg/mL; TGF-beta(2), 377 pg/mL), parapneumonic (TGF-beta(1), 5,024 pg/mL; TGF-beta(2), 464 pg/mL), and miscellaneous exudate groups (TGF-beta(1), 7,690 pg/mL; TGF-beta(2), 369 pg/mL). There was no significant difference in TGF-beta(1) and TGF-beta(2) levels among the four exudate groups. CONCLUSIONS: VEGF levels in pleural effusions are significantly correlated with the levels of TGF-beta(1) and beta(2) isoforms. VEGF, TGF-beta(1), and TGF-beta(2) levels were all higher in exudative effusions than in effusions secondary to CHF.

Ionoregulatory disruption as the acute toxic mechanism for lead in the rainbow trout (Oncorhynchus mykiss).

The mechanism for acute toxicity of lead (Pb) in rainbow trout (Oncorhynchus mykiss) was investigated at Pb concentrations close to the 96 h LC50 of 1.0 mg dissolved Pb l(-1) (0.8-1.4, 95% C.I.) determined in dechlorinated Hamilton city tap water (from Lake Ontario, hardness=140 mg l(-1) CaCO(3)). Tissue Pb accumulation associated with death was highest in the gill, followed by kidney and liver. Significant ionoregulatory impacts were observed in adult rainbow trout (200-300 g) fitted with indwelling dorsal aortic catheters and exposed to 1.1+/-0.04 mg dissolved Pb l(-1). Decreased plasma [Ca(2+)], [Na(+)] and [Cl(-)] occurred after 48 h of exposure through to 120 h, with increases in plasma [Mg(2+)], ammonia, and cortisol. No marked changes in PaO(2), PaCO(2), pH, glucose, or hematological parameters were evident. Branchial Na(+)/K(+) ATPase activity in juvenile trout exposed to concentrations close to the 96 h LC50 was inhibited by approximately 40% after 48 h of Pb exposure. Calcium ion flux measurements using 45Ca as a radiotracer showed 65% inhibition of Ca(2+) influx after 0, 12, 24 or 48 h exposure to the 96 h LC50 concentration of Pb. There was also significant inhibition (40-50%) of both Na(+) and Cl(-) uptake, measured with 22Na and 36Cl simultaneously. We conclude that the mechanism of acute toxicity for Pb in rainbow trout occurs by ionoregulatory disruption rather than respiratory or acid/base distress at Pb concentrations close to the 96 h LC50 in moderately hard water.

Efficacy of Arosurf MSF and formulations of Bacillus thuringiensis var. israelensis against Anopheles albimanus: laboratory bioassay.

The efficacy of Arosurf MSF alone and in combination with three preparations of Bacillus thuringiensis var. israelensis (B.t.i.) against Anopheles albimanus larvae, pupae and eggs was determined by bioassay. Arosurf MSF alone was effective against the egg, 4th larval instar and pupal stages. All Arosurf MSF and B.t.i. combined formulations produced over 90% mortality of all larvae and pupae, 48 hr posttreatment. Egg eclosion was reduced to approximately 25% with all formulations containing Arosurf MSF.

Ferritin translation by interleukin-1and interleukin-6: the role of sequences upstream of the start codons of the heavy and light subunit genes.

Interleukin-1beta (IL-1beta) elevates H- and L-ferritin subunit synthesis in both human hepatoma cells (HepG2) and primary human umbilical vein endothelial cells. Ferritin induction is greater than the increase in total HepG2 protein synthesis in response to IL-1. IL-6 causes a moderate increase in L-subunit synthesis. The levels of the mRNAs for the ferritin H-subunits (H-mRNA) and light subunits (L-mRNA) remain unchanged, indicating that expression of the iron storage protein, ferritin, is regulated by translational mechanisms during inflammation. We have found a translational enhancer region in the L-ferritin mRNA 5'UTR that confers two-fold baseline and twofold IL-1-dependent translational regulation to a CAT reporter message. The L-mRNA motif is related to a 61 nucleotide (nt) G+C-rich translational enhancer within 70 nt of the H-ferritin start codon. Sequences upstream of the start codons (SUS elements) in both H-mRNA and L-mRNAs confer IL-1beta but not IL-6-dependent translation to hybrid ferritin/CAT reporter mRNAs. The H- and L-ferritin mRNA SUS elements contain a motif similar to a consensus reported for the 5' leaders of other acute phase response mRNAs. Transfected hybrid H-mRNA SUS/CAT mRNAs with a three nucleotide deleted version of the H-mRNA SUS displays an eightfold reduced level of translation and no longer confer IL-1beta-dependent translation.